Virus Genes

, Volume 45, Issue 3, pp 440–451 | Cite as

Influence of copy number on the expression levels of pandemic influenza hemagglutinin recombinant protein in methylotrophic yeast Pichia pastoris

  • T. N. AthmaramEmail author
  • Shweta Saraswat
  • Anil Kumar Singh
  • M. Kameswara Rao
  • N. Gopalan
  • V. V. S. Suryanarayana
  • P. V. L. Rao


The hemagglutinin (HA) gene of novel Swine Origin Influenza A/California/04/2009 (H1N1) was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA-synthetic gene having α secretory tag under the control of AOX1 promoter was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having single and multiple copy integrants of the expression cassettes were screened for the expression of full length HA protein in the culture supernatant. In order to completely exploit the expression potential of the P. pastoris expression system, a systematic investigation on the influence of gene copy number on the expression of the recombinant protein was made. A panel of Pichia clones carrying increasing copies of the heterologous gene was selected based on Geneticin resistance and SYBR green-based quantitative real-time PCR approach. Using these strategies, recombinant Pichia transformants carrying up to a maximum of four to six copies of the transgene were identified. After optimising the expression conditions for shaker flask culture, the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the expression level of H1N1HA recombinant protein. Our findings clearly suggest that the gene dosage effect play a vital role in high level expression of the pandemic Influenza HA protein in yeast system.


Pichia pastoris Yeast Copy number, swine origin influenza A virus H1N1 Hemagglutinin Gene dosage effect 



The authors are thankful to Dr. B. K. Bhattacharya, Head, Div. of Biochemistry for his help on MALDI-TOF/MS study. The authors also acknowledge Director, DRDE, Gwalior, Madhya Pradesh-474 002 for his kind support in executing this study.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • T. N. Athmaram
    • 1
    Email author
  • Shweta Saraswat
    • 1
  • Anil Kumar Singh
    • 2
  • M. Kameswara Rao
    • 3
  • N. Gopalan
    • 4
  • V. V. S. Suryanarayana
    • 5
  • P. V. L. Rao
    • 1
  1. 1.Division of Virology, Defence Research and Development EstablishmentMinistry of Defence (Govt. of India)GwaliorIndia
  2. 2.Bioprocess Scale-Up Facility, Defence Research and Development EstablishmentMinistry of Defence (Govt. of India)GwaliorIndia
  3. 3.Division of Biochemistry, Defence Research and Development EstablishmentMinistry of Defence (Govt. of India)GwaliorIndia
  4. 4.Vector Management Division, Defence Research and Development EstablishmentMinistry of Defence (Govt. of India)GwaliorIndia
  5. 5.Molecular Virology LaboratoryIndian Veterinary Research InstituteBengaluruIndia

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